Einleitung. Bei dem Komplementinhibitor Faktor H (FH) mit einem Molekulargewicht von 155 kDa handelt es sich um das dominierende lösliche Regulatorprotein des Komplementsystems. Mit einer Konzentration von 250–600µg/ml in humanem und Rattenserum fungiert FH als Ko-Faktor für die Faktor I-vermittelte Spaltung der Komponente C3b, konkurriert mit Faktor B um die Bindung an C3b und induziert den Zerfall des C3bBb Komplexes. Der monomere FH ist wie andere Komplementinhibitoren aus dem RCA-Cluster modulär aufgebaut. Syntheseorte des FH sind sowohl Hepatozyten als auch Kupfferzellen (KC).
Antibodies directed against HLA antigens of a given donor represent the most prominent cause for hyper-acute and acute rejections. In order to select recipients without donor-specific antibodies the complement-dependent cytotoxicity (CDC-) crossmatch as the standard procedure was established. As a functional assay it strongly depends on the availability of isolated donor lymphocytes and in particular on their vitality. However, due to several diseases or pharmacological treatment of a given recipient unexpected "false-positive" results of the CDC-crossmatch may arise. We here present three groups of patients which demonstrate the limits of the conventional crossmatch. 1) Kidney recipients before living donations exhibited positive CDC-reactions due to their conditioning using the therapeutical anti-CD20 mAb Rituximab (n=7), routinely used to deplete B-cells, or the anti-CD25 mAb basiliximab (n=2) to inhibit the proliferation of activated T-cells. 2) Recipients suffering from various leukaemias (n=5) exhibited "positive" CDC-crossmatches using PBL of the donors, although formerly these patients had never shown anti-HLA antibodies. Instead of donor-specific allo-antibodies, cytostatic agents such as 6-mercaptopurine led to an unspecific cell death. 3) Patients projected for post mortem or living kidney donations (n=44) exhibited "positive" CDC-crossmatch results which were not in accordance with their former antibody status and, partially, with high degrees of HLA-matching. These implausible results were due to underlying auto-immune diseases, mainly of the systemic immune complex type III such as lupus erythematosus, mainly leading to false-positive B-cell crossmatches by immune complexes binding to Fcγ-receptors. In all these 58 cases the alternatively performed ELISA-based "Antibody Monitoring System" (AMS-) crossmatch assay was not artifically affected, suggesting that this assay may be comprehensively established at least for the cases described.
Rat Kupffer cells (KC), hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) all express the C5a receptor (C5aR) constitutively in contrast to hepatocytes (HC). HSC showed an unexpectedly high level of expression of the C5aR. As these cells are known to play a key role in the induction of liver fibrosis we hypothesized that C5a may possibly induce fibrogenetic proteins in these cells. HSC are known to express the extracellular matrix (ECM) proteins collagen IV, fibronectin, entactin and the structure protein smooth muscle actin (SMA) which is regarded as a marker for the fibrotic conversion of HSC to myofibroblast-like cells. We investigated the effect of recombinant rat C5a (rrC5a) on the upregulation of these ECM-proteins and of SMA, all of which are known to be expressed by HSC. The profibrotic cytokine TGF-beta1 (2 ng/ml), which was used as a control, clearly upregulated the three matrix proteins but not SMA. In the absence of any stimulus HSC upregulated the three ECM-proteins as well as SMA during their conversion into myofibroblast-like cells. This resulted in a high stimulus-independent plateau of the mRNA expressions for all four proteins after four to five days of culture. Readouts were therefore taken at 72 h after the isolation of the HSC when the investigated mRNA levels had not yet reached their maxima due to the conversion of the cells. The first 24 h of culture were performed without stimulus and the following 48 h in the presence of 100 nM rrC5a (1 micro g/ml) or TGF-beta1 (2 ng/ml). Only fibronectin-specific mRNA was clearly upregulated by C5a whereas entactin, collagen IV and SMA were not affected by C5a. By competitive-quantitative PCR the upregulation of fibronectin-specific mRNA was determined to be about five-fold. As TGF-beta1 upregulated all of the three investigated ECM-proteins but not SMA it was checked as to whether C5a might act indirectly by upregulating the expression of TGF-beta1 in KC and HSC, as both cell types are known to be sources of this profibrotic cytokine. However, using RT-PCR, such an effect was not detectable in either cell type after 3, 10 or 24 h.
The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and in-vivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-beta1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.
Abstract The human leukocyte antigen (HLA) system is a major part of the human immune system and has an impact on tumor initiation, tumor progression and immunosurveillance. Renal cell carcinoma tumors are considered to be immunogenic. Therefore, we studied the allele frequencies of four gene loci (HLA-A, -B, -C and HLA-DR) in a cohort of 106 German renal cell carcinoma (RCC) patients and in 201 healthy controls. HLA-A-C were determined using serological methods, whereas HLA-C12, C14, C16, C18 and HLA-DR were characterized through the use of standard molecular biological methods. The presence of HLA-A and HLA-B alleles did not differ significantly between RCC patients and healthy controls. However, the occurrence of the HLA-C*12 allele was significantly increased in German RCC patients compared with healthy controls (P < 0.005; RR=2.3; Fisher’s exact test), whereas the occurrence of the HLA-DRB1*04 allele was significantly reduced in RCC patients compared with healthy controls (P < 0.05; RR=0.7; Fisher’s exact test). But the presence of allele HLA-C*12 was not significantly associated with 10 years overall survival. We suggest that the frequency of HLA alleles can affect development of RCC and could add knowledge as predictive marker for future immunotherapies. Citation Format: Steffen Göbel, Astrid Kehlen, Karen Bluemke, Wolfgang Altermann, Gerald Schlaf, Kersten Fischer, Paolo Fornara, Bernd Wullich, Sven Wach, Helge W. Taubert. Frequencies of HLA-Class I and II alleles between German patients with renal cell carcinoma and healthy controls can be different [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5605. doi:10.1158/1538-7445.AM2017-5605
Donorspezifische Anti-HLA-Antikörper stellen die häufigste Ursache für hyperakute bzw. akute Abstoßungsepisoden nach Transplantationen solider Organe dar. Zum Ausschluss dieser Antikörper wurde bereits vor mehr als 40 Jahren der komplementabhängige Lymphozytotoxizitätstest (LZT/engl. CDC) entwickelt. Anhand einer Kasuistik wird dargestellt, wie dieses diagnostische Verfahren, ein funktioneller Vitalitätsassay, als gesetzlich vorgegebene Standardprozedur unter dem Einfluss der Typ-III-Autoimmunerkrankung systemischer Lupus erythematodes (SLE) zu einem falsch positiven Ergebnis führt und somit eine Kontraindikation für eine anstehende Nierentransplantation vortäuschen kann. Zusätzlich werden die plausiblen und validen Ergebnisse des alternativen Festphasen-basierenden Kreuztestverfahrens, das für derartige Artefakte nicht in gleicher Weise empfänglich ist, dargestellt. Kritisch diskutiert wird in diesem Zusammenhang das in den Richtlinien der Bundesärztekammer im Jahr 2010 als verbindlich festgelegte LZT-Kreuztestverfahren vor dem Hintergrund einer Anhäufung von Patienten mit Erkrankung des autoimmunen Formenkreises auf den Wartelisten der Organe, für deren Zuteilung ein negatives Resultat im Prätransplantations-Kreuztest gefordert ist. Dieser Patientengruppe bleiben Organe von postmortalen Spendern aufgrund falsch positiver Kreuztestergebnisse häufig versagt.
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